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Abstract

3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGR) catalyzes the conversion of HMG CoA to mevalonate, which is the rate limiting step in the production of cholesterol and numerous nonsterol isoprenoid products. Mammalian HMGR is regulated by transcriptional and post-transcriptional feedback mechanisms. The transcriptional regulation is mediated by sterol regulatory element binding proteins (SREBPs), which are synthesized as inactive precursors in the endoplasmic reticulum (ER) membrane. In the absence of sterols, SREBP cleavage activating protein (SCAP) escorts SREBPs from ER to the Golgi apparatus, where SREBPs are cleaved by site 1 and site 2 proteases so as to release their amino terminal transcription factor domains to the nucleus. Sterols inhibit the exit of SCAP-SREBP complex from the ER by promoting the binding of two related polytopic ER membrane proteins, Insig-1 and Insig-2, to the membrane domain of SCAP. Insig-1, but not Insig-2, is an SREBP target gene, causing Insig-1 levels to drop in the presence of sterols, when it is expected to exert its action. The degradation of HMGR requires both sterols and a nonsterol product of the mevalonate pathway and the eight membrane spanning segments in its amino terminus. The membrane domains of HMGR and SCAP bear sequence similarity prompting the investigation of whether Insig proteins can also bind to HMGR. Indeed, Insig-1 and Insig-2 were found to interact with HMGR in a regulated manner and mediate its proteasomal degradation. This effect can be specifically inhibited by overexpressing the membrane domain of SCAP. Insigs were shown to promote the ubiquitination of HMGR on lysine 248 in the cytoplasmic loop between transmembrane segments 6 and 7. In an attempt to achieve a better understanding of the mechanism by which HMGR is degraded, a genetic approach was developed to select mutant somatic cells that cannot degrade HMGR in the presence of sterols. The isolation and characterization of Chinese hamster ovary cells deficient in Insig-1 confirmed the endogenous requirement of Insig-1 for HMGR degradation and revealed the role of differential regulation of Insig-1 and Insig-2 in terms of SREBP processing. These studies revealed a complex feedback regulatory system governing cholesterol homeostasis.